The vasculature forms by a combination of vasculogenesis and angiogenesis. Convincing data indicate a role for vascular endothelial growth factor (VEGF) in both process. VEGF exists as three isoforms that arise by alternative splicing. Recent data suggest that the isoforms differ in their mitogenic potential but other functional differences have yet to be determined. In addition, the precise role and regulation of VEGF during vascular development are known. Finally, little is known about the molecules that maintain the adult vasculature in a differentiated, quiescent state. This proposal is founded on the hypothesis that specific molecules regulate vascular growth and differentiation. Our long-term objective is to identify these molecules and their functions. To accomplish this following aims are proposed. (1) To examine the expression and function of the VEGF isoforms during development in vivo. Isoform expression will be examined during mouse development using Rnase protection and in situ hybridization. Isoform function will be assessed by targeted exon deletion to generate mice expression specific VEGF isoforms. (2) To investigate the role and regulation of VEGF during vascular development. ES cells differentiated into embryoid bodies will be used as an in vitro model of vasculogenesis. The regulation of VEGF expression will be characterized in this model by studying the expression of stably transfected VEGF promoter/reporter constructs. VEGF isoform function will also be examined in this system. (3) To characterize the nature and function of a novel gene highly expressed by quiescent but not proliferating EC. This gene (frizzled) is homologous in C. elegans through humans. Expression studies in vivo and in vitro suggest the gene product may contribute to the regulation of endothelial growth and/or differentiation. Together the results of these investigations should add to our understanding of the mechanism (s) that regulate vascular growth and differentiation.